Lenka Záveská Drábková

Comparison of seven DNA extraction and amplification protocols in historical herbarium specimens of juncaceae

Seven DNA extraction protocols were used to obtain DNA from herbarium specimens ofJuncus andLuzula (Juncaceae) of various ages. DNA of historical samples is difficult to extract, and the extracts are seldom of good quality. The quality of DNA obtained was estimated by using a spectrophotometer to measure the A260/280 absorbance ratio. The total DNA yield was measured by a fluorometer. The results indicate the success of using both mixer mill grinding and a DNeasy Plant Kit. Another extraction protocol (grinding with mortar and pestle, using liquid nitrogen) yielded DNA from many samples. Modified CTAB extraction, with a lengthy precipitation, usually provided good amounts of DNA. Other protocols did not give satisfactory results.

Phylogeny of the Juncaceae based on rbcL sequences, with special emphasis on Luzula DC. and Juncus L.

Abstract.Cladistic analysis of rbcL nucleotide sequences was applied to 58 taxa representing most subgenera and sections of Luzula and Juncus, chosen to reflect morphological and geographical diversity of both genera. Additionally, representatives of all other genera of the Juncaceae and two taxa from the Cyperaceae were included. Phylogenetic trees were constructed using parsimony with Prioniumserratum as outgroup. The dataset has 190 parsimony informative sites. The analysis yielded more than 332,400 equally parsimonious trees (length 620, CI=0.47, RI=0.82). A jackknife analysis revealed several well-supported clades. Luzula is monophyletic and Juncus is non-monophyletic. Each of the generally accepted subgenera of Juncus, subg. Juncus and subg. Agathryon, form a clade, but their circumscription differs from the traditional views. The subgenera recognized in Luzula remain mainly unresolved. A well-supported clade is represented by an assemblage of representatives of five genera and species distributed in the Southern Hemisphere: Juncuscapensis and J. lomatophyllus (both from section Graminifolii), Rostkovia, Distichia, Marsippospermum, and Patosia.

Phytohormone Profiling across the Bryophytes

Background Bryophytes represent a very diverse group of non-vascular plants such as mosses, liverworts and hornworts and the oldest extant lineage of land plants. Determination of endogenous phytohormone profiles in bryophytes can provide substantial information about early land plant evolution. In this study, we screened thirty bryophyte species including six liverworts and twenty-four mosses for their phytohormone profiles in order to relate the hormonome with phylogeny in the plant kingdom. Methodology Samples belonging to nine orders (Pelliales, Jungermanniales, Porellales, Sphagnales, Tetraphidales, Polytrichales, Dicranales, Bryales, Hypnales) were collected in Central and Northern Bohemia. The phytohormone content was analysed with a high performance liquid chromatography electrospray tandem-mass spectrometry (HPLC-ESI-MS/MS). Principal Findings As revealed for growth hormones, some common traits such as weak conjugation of both cytokinins and auxins, intensive production of cisZ-type cytokinins and strong oxidative degradation of auxins with abundance of a major primary catabolite 2-oxindole-3-acetic acid were pronounced in all bryophytes. Whereas apparent dissimilarities in growth hormones profiles between liverworts and mosses were evident, no obvious trends in stress hormone levels (abscisic acid, jasmonic acid, salicylic acid) were found with respect to the phylogeny. Conclusion The apparent differences in conjugation and/or degradation strategies of growth hormones between liverworts and mosses might potentially show a hidden link between vascular plants and liverworts. On the other hand, the complement of stress hormones in bryophytes probably correlate rather with prevailing environmental conditions and plant survival strategy than with plant evolution.

Analysis of nrDNA polymorphism in closely related diploid sexual, tetraploid sexual and polyploid agamospermous species

Nuclear sequences of ITS1-5.8S-ITS2 region of rDNA may be an important source of phylogenetically informative data provided that nrDNA is cloned and the character of sequence variation of clones is properly analyzed. nrDNA of selected Taraxacum sections was studied to show sequence variation differences among diploid sexual, tetraploid sexual and polyploid agamospermous species. We examined nucleotide characteristics, substitution pattern, secondary structure, and the phylogenetic utility of ITS1-5.8S-ITS2 from 301 clones of 32 species representing 11 sections. The most divergent sequences of ITS1&2 differed by 17.1% and in 5.8S only by 3.7%. The ITS1-5.8S-ITS2 characteristics, integrity and also stability of secondary structures confirmed that pseudogenes are not responsible for the above variation. The within-individual polymorphism of clones implies that the concerted evolution of ITS cistron of agamospermous polyploid Taraxacum is remarkably suppressed. Sequences of ITS clones proved to be a useful tool for mapping pathways of complex reticulation (polyploid hybridity) in agamospermous Taraxacum.

Evaluation of Cell-Free Urine microRNAs Expression for the Use in Diagnosis of Ovarian and Endometrial Cancers. A Pilot Study

Among gynaecological cancers, epithelial ovarian cancers are the most deadly cancers while endometrial cancers are the most common diseases. Efforts to establish relevant novel diagnostic, screening and prognostic markers are aimed to help reduce the high level of mortality, chemoresistance and recurrence, particularly in ovarian cancer. MicroRNAs, the class of post-transcriptional regulators, have emerged as the promising diagnostic and prognostic markers associated with various diseased states recently. Urine has been shown as the source of microRNAs several years ago; however, there has been lack of information on urine microRNA expression in ovarian and endometrial cancers till now. In this pilot study, we examined the expression of candidate cell-free urine microRNAs in ovarian cancer and endometrial cancer patients using quantitative real-time PCR. We compared the expression between pre- and post-surgery ovarian cancer samples, and between patients with ovarian and endometrial cancers and healthy controls, within three types of experiments. These experiments evaluated three different isolation methods of urine RNA, representing two supernatant and one exosome fractions of extracellular microRNA. In ovarian cancer, we found miR-92a significantly up-regulated, and miR-106b significantly down-regulated in comparison with control samples. In endometrial cancer, only miR-106b was found down-regulated significantly compared to control samples. Using exosome RNA, no significant de-regulations in microRNAs expression could be found in either of the cancers investigated. We propose that more research should now focus on confirming the diagnostic potential of urine microRNAs in gynaecological cancers using more clinical samples and large-scale expression profiling methods.

Molecular phylogeny of the genus Luzula DC. (Juncaceae, Monocotyledones) based on plastome and nuclear ribosomal regions: a case of incongruence, incomplete lineage sorting and hybridisation.

The genus Luzula consists of 115 species distributed throughout the world. Luzula is monophyletic, but species relationships within the genus are difficult to determine primarily due to the similar morphology even within geographically remote taxa (especially within the section Luzula). The plastome trnL intron, trnL-F intergenic spacer and the nuclear ribosomal ITS1-5.8S-ITS2 regions were analysed using maximum parsimony and maximum likelihood reconstruction in 93 species of Luzula. The incongruent phylogenetic signals obtained from the chloroplast and the nuclear genomes point to incomplete lineage sorting as well as recent hybridisation in this group. Although tree-building analyses revealed several well-supported lineages, the outcomes for many groups were ambiguous. In the total evidence tree, Luzula species were grouped within six main clades (1. subgenus Marlenia, 2. subgenus Pterodes except for L. pilosa, 3. sections Anthelaea and Nodulosae, 4. sections Diprophyllatae and Thyrsanochlamydeae, 5. section Alpinae except for a few species and 6. section Luzula). The subgenus Marlenia occupies the early derived lineage within the genus Luzula. The traditionally accepted subgenera Pterodes and Luzula (and its sections) appear to be non-monophyletic. A statistical parsimony network approach showed that ancient haplotypes and ribotypes co-occur with their descendants in Luzula. Furthermore, many haplotypes are shared among different species. Within the Luzula section Luzula, both recent hybridisation and incomplete lineage sorting of ancestral polymorphisms may represent potential sources of the incongruence between chloroplast and nuclear data.

DNA Taxonomy—the Riddle of Oxychloë (Juncaceae)

Abstract Recently, advocates of DNA taxonomy have complained that there is inadequate control of the taxonomy in databases such as GenBank. This is correct, but the uncertainty may be extended to the sequences themselves. The present study shows that as long as vouchers are available neither problem is fatal, but if no voucher exists, bad sequences and bad taxonomy may be forever linked. Previous phylogenetic analyses of rbcL sequences have indicated that the small, south hemisphere, genus Oxychloë (Juncaceae) surprisingly either is embedded within or is a sister group to the Cyperaceae. This is not in accordance with traditional or current morphological data. By studying five new accessions, representing four species of Oxychloë, and re-examining the two vouchers of O. andina that were used in previous phylogenies, it has been possible to show that these two sequences are erroneous. One is a chimeric sequence and the other is most likely “a contaminant.”

Phylogenetic relationships within Luzula DC. and Juncus L. (Juncaceae): A comparison of phylogenetic signals of trnL‐trnF intergenic spacer, trnL intron and rbcL plastome sequence data

Juncus and Luzula are the largest, almost cosmopolitan, genera in the Juncaceae. Relationships within Juncus and Luzula and among other genera of Juncaceae (Distichia, Marsippospermum, Oxychloë, Patosia and Rostkovia) remain incompletely resolved. RbcL sequence data resolved a part of the supraspecific phylogeny, but many clades remain polytomic. For this reason, the non‐coding cpDNA regions, trnL intron and trnL‐trnF intergenic spacer, were sequenced. We intended to create hypotheses of relationships within Juncaceae and to test the classification of the sections, but a primary goal to this study was to assess the relationships within Juncus and Luzula and to test for monophyly of groups recognized from rbcL data (especially the monophyly of genus Luzula and the Southern Hemisphere Clade (SHC)). Furthermore, we tested the influence of different rooting and ingroup composition on the tree topology. The parsimony analyses revealed several well‐supported lineages. The traditionally distinguished genus Luzula is monophyletic and Juncus is non‐monophyletic. Two subgenera of Luzula (Pterodes and Luzula) are non‐monophyletic, while subg. Marlenia forms a sister group to the whole Luzula clade (trnL‐F data set). Within Juncus, both subgenus Juncus and subgenus Agathryon are non‐monophyletic. SHC is clustered not only with the South African J. lomatophylus and J. capensis, but also together with members of the section Juncus, Caespitosi and Graminifolii. These sections form a well‐separated sister group to the SHC. Within the genera Juncus and Luzula, monophyly is demonstrated for a number of groups (e.g., Juncus section Stygiopsis, Luzula section Luzula) but questioned for others (e.g., Juncus section Graminifolii). The unusual, separate positioning of Juncus trifidus and J. monanthos were clarified by trnL‐trnF sequence data, but vary within the tree topology depending on outgroup selection and also due to LBA phenomenon.

Control of cytokinin and auxin homeostasis in cyanobacteria and algae

Background and Aims The metabolism of cytokinins (CKs) and auxins in vascular plants is relatively well understood, but data concerning their metabolic pathways in non-vascular plants are still rather rare. With the aim of filling this gap, 20 representatives of taxonomically major lineages of cyanobacteria and algae from Cyanophyceae, Xanthophyceae, Eustigmatophyceae, Porphyridiophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Zygnematophyceae and Klebsormidiophyceae were analysed for endogenous profiles of CKs and auxins and some of them were used for studies of the metabolic fate of exogenously applied radiolabelled CK, [3H]trans-zeatin (transZ) and auxin ([3H]indole-3-acetic acid (IAA)), and the dynamics of endogenous CK and auxin pools during algal growth and cell division. Methods Quantification of phytohormone levels was performed by high-performance or ultrahigh-performance liquid chromatography–electrospray tandem mass spectrometry (HPLC-MS/MS, UHPLC-MS/MS). The dynamics of exogenously applied [3H]transZ and [3H]IAA in cell cultures were monitored by HPLC with on-line radioactivity detection. Key Results The comprehensive screen of selected cyanobacteria and algae for endogenous CKs revealed a predominance of bioactive and phosphate CK forms while O- and N-glucosides evidently did not contribute greatly to the total CK pool. The abundance of cis-zeatin-type CKs and occurrence of CK 2-methylthio derivatives pointed to the tRNA pathway as a substantial source of CKs. The importance of the tRNA biosynthetic pathway was proved by the detection of tRNA-bound CKs during the course of Scenedesmus obliquus growth. Among auxins, free IAA and its oxidation catabolite 2-oxindole-3-acetic acid represented the prevailing endogenous forms. After treatment with [3H]IAA, IAA-aspartate and indole-3-acetyl-1-glucosyl ester were detected as major auxin metabolites. Moreover, different dynamics of endogenous CKs and auxin profiles during S. obliquus culture clearly demonstrated diverse roles of both phytohormones in algal growth and cell division. Conclusions Our data suggest the existence and functioning of a complex network of metabolic pathways and activity control of CKs and auxins in cyanobacteria and algae that apparently differ from those in vascular plants.

Towards a better understanding of the Taraxacum evolution (Compositae–Cichorieae) on the basis of nrDNA of sexually reproducing species

The genus Taraxacum is characterized by prevailing complex multiple hybridity, frequent polyploidy and widespread agamospermous reproduction, which makes the phylogenetic analysis difficult. On the basis of the previous analysis of the variation of nrDNA in Taraxacum taxa with different ploidy levels and modes of reproduction, to mitigate consequences of the reticulate complexity of the genus, a phylogenetic study of 52 samples of sexually reproducing dandelions of 26 sections (and another 13 agamospermous representatives of other sections known to include sexuals) was carried out. Both sexual and agamospermous samples were analysed using maximum parsimony and neighbour network. Exclusively sexual dandelions were analysed using the same approaches. In spite of the general agreement among various types of analyses, there is a limited overall congruence between results of nrDNA analyses and the established taxonomic system of the genus Taraxacum. The analyses shed light on the relationships among the most primitive groups. A stable clade is formed by representatives of the sections Primigenia, Orientalia, Sonchidium, Piesis and T. cylleneum. Another case of stable relationships is that of the members of the sect. Dioszegia. Relationships between the sects. Erythrosperma and Erythrocarpa were supported, and the relatedness of the members of sect. Australasica was confirmed. Rather unexpectedly, the agamospermous samples of the sect. Oligantha (T. minutilobum) are shown to be closely related with the sect. Macrocornuta. The latter section is generally considered to be close to sect. Ceratoidea (T. koksaghyz) on morphological grounds but this presumption is not corroborated by the results of nrDNA analyses. Analyses of 72 samples of sexual dandelions were also performed using the trnL–trnF region of the chloroplast DNA. The maximum parsimony analysis of this region reveals intraspecific variation in a number of ancestral diploid sexual species, all present in the two main branches of the cladogram. This phenomenon is attributed to the ancient gene flow and possibly to the persistence of ancestral cpDNA polymorphism. The strict consensus cpDNA tree information content and interpretability is quite low. The maximum parsimony analysis of combined nrDNA and cpDNA data sets was also performed with expectably low interpretability of the results.